PROJECT SUMMARY Very little is known of human placental development in the period between one and five weeks of gestation when trophoblasts invade the uterus, form the primitive syncytium and cytotrophoblast, and then primary villi. Thus, models are needed to study the molecular and cellular mechanisms controlling early human placental development and what can go wrong with these processes to cause placental disease and early conceptus loss. It is now well established that human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can be driven along the trophoblast lineage by exposing them to BMP4 and inhibiting the signaling pathways that maintain the pluripotent phenotype. The overall premise is that this in vitro system is a valuable model for mimicking placental trophoblast formed early in the first trimester of pregnancy when it is most vulnerable to many of the same hazards that threaten an in vivo pregnancy early in its existence. The project will use this stem cell model of early trophoblast development to understand the development of the placenta in the earliest stages of pregnancy and the immediate pathological basis of early onset preeclampsia (EOPE), a disease linked to shallow placentation and insufficient perfusion with maternal blood. There are three aims: 1) Test the hypothesis that villous TB from the first half of the first trimester represents a transitional state between the primitive placenta encountered at implantation and the mature placenta of the second and third trimester. The goal is to validate the notion that the ?primitive? TB generated from ESC and iPSC is an in vitro equivalent of early placental TB. Experiments will also confirm preliminary observations that first trimester villous TB shares many features of its molecular signature with both this primitive TB derived from pluripotent cells and more mature placental TB from the second and third trimesters. 2) Employ the stem cell model of trophoblast differentiation to test the hypothesis that stress response pathways are already aberrant in EOPE placentas upon initial formation of trophoblast. The goal is to employ RNAseq analysis and DNA methylation profiling to compare gene and gene network changes associated with PE and CTL cells when they are cultured under normal and stressful oxygen conditions. 3) Test the hypothesis that a better representation of trophoblast emergence will be gained by conducting the differentiation of the pluripotent stem cells to trophoblast with cultured spheroids rather than in 2D-cultures. The plan is to use such a system to follow the emergence of villous trophoblast within organoids. Additionally, trophoblast stem cells (TSC) will be generated from ESC and iPSC and also use these along with ESC/iPSC to create chimeric organoids. Finally the fate of these organoids will be examined when they are placed under the mammary fat pads and kidney capsules of immunocompromised mice to determine whether they exhibit physiologic and structural interactions and invasive potential within the host mouse. A longer term goal will be to determine whether PE and CTL organoids differ in invasiveness at ectopic sites and in interactions with maternal decidualized endometrium.